A Single Amino Acid Substitution in RFC4 Leads to Endoduplication and Compromised Resistance to DNA Damage in Arabidopsis thaliana

Replication factor C (RFC) is a heteropentameric ATPase associated with the diverse cellular activities (AAA+ATPase) protein complex, which is composed of one large subunit, known as RFC1, and four small subunits, RFC2/3/4/5. Among them, RFC1 and RFC3 were previously reported to mediate genomic stability and resistance to pathogens in Arabidopsis. Here, we generated a viable rfc4e (rfc4−1/RFC4(G54E)) mutant with a single amino acid substitution by site-directed mutagenesis. Three of six positive T2 mutants with the same amino acid substitution, but different insertion loci, were sequenced to identify homozygotes, and the three homozygote mutants showed dwarfism, early flowering, and a partially sterile phenotype. RNA sequencing revealed that genes related to DNA repair and replication were highly upregulated. Moreover, the frequency of DNA lesions was found to be increased in rfc4e mutants. Consistent with this, the rfc4e mutants were very sensitive to DSB-inducing genotoxic agents. In addition, the G54E amino acid substitution in AtRFC4 delayed cell cycle progression and led to endoduplication. Overall, our study provides evidence supporting the notion that RFC4 plays an important role in resistance to genotoxicity and cell proliferation by regulating DNA damage repair in Arabidopsis thaliana.